Loratadine is an anti-inflammatory agent against C. difficile toxin B.

BACKGROUND: Clostridium difficile infection (CDI) is a debilitating nosocomial infection. C. difficile produces toxins A and B, which cause inflammation. Existing therapies have issues with recurrence, cost, and safety. We aim to discover a safe, effective, and economical non-microbiological therapeutic approach against CDI. METHODS: We included human primary peripheral blood mononuclear cells (PBMCs), fresh human colonic explants, and humanized HuCD34-NCG mice. Toxin A+B+ VPI10463 and A-B+ ribotype 017 C. difficile strains were used. We used single-cell RNA profiling and high-throughput screening to find actionable toxin B-dependent pathways in PBMCs. RESULTS: Histamine 1 receptor-related drugs were found among the hit compounds that reversed toxin-mediated macrophage inflammatory protein one alpha (MIP-1α) expression in PBMCs. We identified Loratadine as the safest representative antihistamine for therapeutic development. Loratadine inhibited toxin B-induced MIP-1α secretion in fresh human colonic tissues. Oral Loratadine (10 mg/kg/day) maintained survival, inhibited intestinal Ccl3 mRNA expression, and prevented vancomycin-associated recurrence in the VPI10463-infected mice and ribotype 017-infected hamsters. Splenocytes from Loratadine-treated mice conferred anti-inflammatory effects to the VPI10463-infected T/B cell-deficient Rag-/- mice. Oral Loratadine suppressed human MIP-1α expression in monocytes/macrophages in toxin B-expressing ribotype 017-infected humanized HuCD34-NCG mice. CONCLUSIONS: Loratadine may be repurposed to optimize existing therapies against CDI.

Loratadine is an FDA-approved antihistamine that inhibits toxin B-mediated pro-inflammatory macrophage inflammatory protein one alpha secretion from immune cells. The anti-inflammatory effect of Loratadine ameliorates intestinal inflammation in C. difficile-infected animals. Loratadine may be repurposed to optimize existing therapies.

Genistein Inhibits Clostridioides difficile Infection via Estrogen Receptors and Lysine-Deficient Protein Kinase 1.

BACKGROUND: Clostridioides difficile infection (CDI) is a debilitating nosocomial disease. Postmenopausal women may have an increased risk of CDI, suggesting estrogen influence. Soybean products contain a representative estrogenic isoflavone, genistein. METHODS: The anti-inflammatory and antiapoptotic effects of genistein were determined using primary human cells and fresh colonic tissues. The effects of oral genistein therapy among mice and hamsters were evaluated. RESULTS: Within 10 days of CDI, female c57BL/6J mice in a standard environment (regular diet) had a 50% survival rate, while those with estrogen depletion and in an isoflavone-free environment (soy-free diet) had a 25% survival rate. Oral genistein improved their 10-day survival rate to 100% on a regular diet and 75% in an isoflavone-free environment. Genistein reduced macrophage inflammatory protein-1α (MIP-1α) secretion in fresh human colonic tissues exposed to toxins. Genistein inhibited MIP-1α secretion in primary human peripheral blood mononuclear cells, abolished apoptosis and BCL-2-associated X (BAX) expression in human colonic epithelial cells, and activated lysine-deficient protein kinase 1 (WNK1) phosphorylation in both cell types. The anti-inflammatory and antiapoptotic effects of genistein were abolished by inhibiting estrogen receptors and WNK1. CONCLUSIONS: Genistein reduces CDI disease activity by inhibiting proinflammatory cytokine expression and apoptosis via the estrogen receptor/G-protein estrogen receptor/WNK1 pathways.

ADS024, a single-strain live biotherapeutic product of Bacillus velezensis alleviates dextran sulfate-mediated colitis in mice, protects human colonic epithelial cells against apoptosis, and maintains epithelial barrier function.

Epithelial cell apoptosis and compromised gut barrier function are features of inflammatory bowel disease. ADS024 is a single-strain live biotherapeutic product (LBP) of Bacillus velezensis under development for treating ulcerative colitis (UC). The cytoprotective effects of the sterile filtrate of ADS024's secreted products on UC patient-derived colonic tissues, human primary colonic epithelial cells (HPEC), and human colonic epithelial T84 cells were evaluated. ADS024 filtrate significantly inhibited apoptosis and inflammation with reduced Bcl-2 Associated X-protein (BAX) and tumor necrosis factor (TNF) mRNA expression in fresh colonic explants from UC patients. Exposure to UC patient-derived serum exosomes (UCSE) induced apoptosis with increased cleaved caspase 3 protein expression in HPECs. ADS024 filtrate diminished the UCSE-mediated apoptosis by inhibiting cleaved caspase 3. TNFα and interferon-gamma (IFNγ) damaged epithelial barrier integrity with reduced transepithelial electrical resistance (TEER). ADS024 filtrate partially attenuated the TEER reduction and restored tight junction protein 1 (TJP1) expression. Oral live ADS024 treatment reduced weight loss, disease activity, colonic mucosal injury, and colonic expression of interleukin 6 (IL-6) and TNFα in dextran sodium sulfate (DSS)-treated mice with colitis. Thus, ADS024 may protect the colonic epithelial barrier in UC via anti-inflammatory, anti-apoptotic, and tight-junction protection mechanisms.

Elafin Reverses Intestinal Fibrosis by Inhibiting Cathepsin S-Mediated Protease-Activated Receptor 2.

BACKGROUND & AIMS: More than half of Crohn's disease patients develop intestinal fibrosis-induced intestinal strictures. Elafin is a human protease inhibitor that is down-regulated in the stricturing intestine of Crohn's disease patients. We investigated the efficacy of elafin in reversing intestinal fibrosis and elucidated its mechanism of action. METHODS: We developed a new method to mimic a stricturing Crohn's disease environment and induce fibrogenesis using stricturing Crohn's disease patient-derived serum exosomes to condition fresh human intestinal tissues and primary stricturing Crohn's disease patient-derived intestinal fibroblasts. Three mouse models of intestinal fibrosis, including SAMP1/YitFc mice, Salmonella-infected mice, and trinitrobenzene sulfonic acid-treated mice, were also studied. Elafin-Eudragit FS30D formulation and elafin-overexpressing construct and lentivirus were used. RESULTS: Elafin reversed collagen synthesis in human intestinal tissues and fibroblasts pretreated with Crohn's disease patient-derived serum exosomes. Proteome arrays identified cathepsin S as a novel fibroblast-derived pro-fibrogenic protease. Elafin directly suppressed cathepsin S activity to inhibit protease-activated receptor 2 activity and Zinc finger E-box-binding homeobox 1 expression, leading to reduced collagen expression in intestinal fibroblasts. Elafin overexpression reversed ileal fibrosis in SAMP1/YitFc mice, cecal fibrosis in Salmonella-infected mice, and colonic fibrosis in trinitrobenzene sulfonic acid-treated mice. Cathepsin S, protease-activated receptor 2 agonist, and zinc finger E-box-binding homeobox 1 overexpression abolished the anti-fibrogenic effect of elafin in fibroblasts and all 3 mouse models of intestinal fibrosis. Oral elafin-Eudragit FS30D treatment abolished colonic fibrosis in trinitrobenzene sulfonic acid-treated mice. CONCLUSIONS: Elafin suppresses collagen synthesis in intestinal fibroblasts via cathepsin S-dependent protease-activated receptor 2 inhibition and decreases zinc finger E-box-binding homeobox 1 expression. The reduced collagen synthesis leads to the reversal of intestinal fibrosis. Thus, modified elafin may be a therapeutic approach for intestinal fibrosis.

Mice expressing fluorescent PAR2 reveal that endocytosis mediates colonic inflammation and pain.

G protein-coupled receptors (GPCRs) regulate many pathophysiological processes and are major therapeutic targets. The impact of disease on the subcellular distribution and function of GPCRs is poorly understood. We investigated trafficking and signaling of protease-activated receptor 2 (PAR2) in colitis. To localize PAR2 and assess redistribution during disease, we generated knockin mice expressing PAR2 fused to monomeric ultrastable green fluorescent protein (muGFP). PAR2-muGFP signaled and trafficked normally. PAR2 messenger RNA was detected at similar levels in Par2-mugfp and wild-type mice. Immunostaining with a GFP antibody and RNAScope in situ hybridization using F2rl1 (PAR2) and Gfp probes revealed that PAR2-muGFP was expressed in epithelial cells of the small and large intestine and in subsets of enteric and dorsal root ganglia neurons. In healthy mice, PAR2-muGFP was prominently localized to the basolateral membrane of colonocytes. In mice with colitis, PAR2-muGFP was depleted from the plasma membrane of colonocytes and redistributed to early endosomes, consistent with generation of proinflammatory proteases that activate PAR2 PAR2 agonists stimulated endocytosis of PAR2 and recruitment of Gαq, Gαi, and ß-arrestin to early endosomes of T84 colon carcinoma cells. PAR2 agonists increased paracellular permeability of colonic epithelial cells, induced colonic inflammation and hyperalgesia in mice, and stimulated proinflammatory cytokine release from segments of human colon. Knockdown of dynamin-2 (Dnm2), the major colonocyte isoform, and Dnm inhibition attenuated PAR2 endocytosis, signaling complex assembly and colonic inflammation and hyperalgesia. Thus, PAR2 endocytosis sustains protease-evoked inflammation and nociception and PAR2 in endosomes is a potential therapeutic target for colitis.

ADS024, a Bacillus velezensis strain, protects human colonic epithelial cells against C. difficile toxin-mediated apoptosis.

Clostridioides difficile infection (CDI) causes intestinal injury. Toxin A and toxin B cause intestinal injury by inducing colonic epithelial cell apoptosis. ADS024 is a Bacillus velezensis strain in development as a single-strain live biotherapeutic product (SS-LBP) to prevent the recurrence of CDI following the completion of standard antibiotic treatment. We evaluated the protective effects of the sterile filtrate and ethyl acetate extract of conditioned media from ADS024 and DSM7 (control strain) against mucosal epithelial injury in toxin-treated human colonic tissues and apoptosis in toxin-treated human colonic epithelial cells. Ethyl acetate extracts were generated from conditioned culture media from DSM7 and ADS024. Toxin A and toxin B exposure caused epithelial injury in fresh human colonic explants. The sterile filtrate of ADS024, but not DSM7, prevented toxin B-mediated epithelial injury in fresh human colonic explants. Both sterile filtrate and ethyl acetate extract of ADS024 prevented toxin-mediated apoptosis in human colonic epithelial cells. The anti-apoptotic effects of ADS024 filtrate and ethyl acetate extract were dependent on the inhibition of caspase 3 cleavage. The sterile filtrate, but not ethyl acetate extract, of ADS024 partially degraded toxin B. ADS024 inhibits toxin B-mediated apoptosis in human colonic epithelial cells and colonic explants.

Elafin inhibits obesity, hyperglycemia, and liver steatosis in high-fat diet-treated male mice.

Elafin is an antimicrobial and anti-inflammatory protein. We hypothesize that elafin expression correlates with diabetes. Among non-diabetic and prediabetic groups, men have significantly higher serum elafin levels than women. Men with type 2 diabetes mellitus (T2DM) have significantly lower serum elafin levels than men without T2DM. Serum elafin levels are inversely correlated with fasting blood glucose and hemoglobin A1c levels in men with T2DM, but not women with T2DM. Lentiviral elafin overexpression inhibited obesity, hyperglycemia, and liver steatosis in high-fat diet (HFD)-treated male mice. Elafin-overexpressing HFD-treated male mice had increased serum leptin levels, and serum exosomal miR181b-5p and miR219-5p expression. Transplantation of splenocytes and serum exosomes from elafin-overexpressing HFD-treated donor mice reduced food consumption and fat mass, and increased adipose tissue leptin mRNA expression in HFD-treated recipient mice. Elafin improved leptin sensitivity via reduced interferon-gamma expression and induced adipose leptin expression via increased miR181b-5p and miR219-5p expression. Subcutaneous and oral administration of modified elafin inhibited obesity, hyperglycemia, and liver steatosis in the HFD-treated mice. Circulating elafin levels are associated with hyperglycemia in men with T2DM. Elafin, via immune-derived miRNAs and cytokine, activates leptin sensitivity and expression that subsequently inhibit food consumption, obesity, hyperglycemia, and liver steatosis in HFD-treated male mice.

High circulating elafin levels are associated with Crohn's disease-associated intestinal strictures.

BACKGROUND: Antimicrobial peptide expression is associated with disease activity in inflammatory bowel disease (IBD) patients. IBD patients have abnormal expression of elafin, a human elastase-specific protease inhibitor and antimicrobial peptide. We determined elafin expression in blood, intestine, and mesenteric fat of IBD and non-IBD patients. METHODS: Serum samples from normal and IBD patients were collected from two UCLA cohorts. Surgical resection samples of human colonic and mesenteric fat tissues from IBD and non-IBD (colon cancer) patients were collected from Cedars-Sinai Medical Center. RESULTS: High serum elafin levels were associated with a significantly elevated risk of intestinal stricture in Crohn's disease (CD) patients. Microsoft Azure Machine learning algorithm using serum elafin levels and clinical data identified stricturing CD patients with high accuracy. Serum elafin levels had weak positive correlations with clinical disease activity (Partial Mayo Score and Harvey Bradshaw Index), but not endoscopic disease activity (Mayo Endoscopic Subscore and Simple Endoscopic Index for CD) in IBD patients. Ulcerative colitis (UC) patients had high serum elafin levels. Colonic elafin mRNA and protein expression were not associated with clinical disease activity and histological injury in IBD patients, but stricturing CD patients had lower colonic elafin expression than non-stricturing CD patients. Mesenteric fat in stricturing CD patients had significantly increased elafin mRNA and protein expression, which may contribute to high circulating elafin levels. Human mesenteric fat adipocytes secrete elafin protein. CONCLUSIONS: High circulating elafin levels are associated with the presence of stricture in CD patients. Serum elafin levels may help identify intestinal strictures in CD patients.

Therapeutic Mechanism of Macrophage Inflammatory Protein 1 α Neutralizing Antibody (CCL3) in Clostridium difficile Infection in Mice.

BACKGROUND: Clostridium difficile infection (CDI) causes diarrhea and colitis. We aimed to find a common pathogenic pathway in CDI among humans and mice by comparing toxin-mediated effects in human and mouse colonic tissues. METHOD: Using multiplex enzyme-linked immunosorbent assay, we determined the cytokine secretion of toxin A- and B-treated human and mouse colonic explants. RESULTS: Toxin A and toxin B exposure to fresh human and mouse colonic explants caused different patterns of cytokine secretion. Toxin A induced macrophage inflammatory protein (MIP) 1α secretion in both human and mouse explants. Toxin A reduced the expression of chloride anion exchanger SLC26A3 expression in mouse colonic explants and human colonic epithelial cells. Patients with CDI had increased colonic MIP-1 α expression and reduced colonic SLC26A3 (solute carrier family 26, member 3) compared with controls. Anti-MIP-1 α neutralizing antibody prevented death, ameliorated colonic injury, reduced colonic interleukin 1ß (IL-1ß) messenger RNA expression, and restored colonic SLC26a3 expression in C. difficile-infected mice. The anti-MIP-1 α neutralizing antibody prevented CDI recurrence. SLC26a3 inhibition augmented colonic IL-1 ß messenger RNA expression and abolished the protective effect of anti-MIP-1 α neutralizing antibody in mice with CDI. CONCLUSION: MIP-1 α is a common toxin A-dependent chemokine in human and mouse colon. MIP-1 α mediates detrimental effects by reducing SLC26a3 and enhancing IL-1 ß expression in the colon.

Cathelicidin Suppresses Colon Cancer Metastasis via a P2RX7-Dependent Mechanism.

The antimicrobial peptide cathelicidin inhibits development of colitis-associated colon cancer. However, the role of cathelicidin in colon cancer metastasis remains unknown. We hypothesized that cathelicidin is effective in inhibiting colon cancer metastasis. Human colon cancer HT-29 cells were injected intravenously into nude mice. Control HA-tagged adeno-associated virus (HA-AAV) or cathelicidin-overexpressing AAV (CAMP-HA-AAV) were injected intravenously into nude mice on the same day. Four weeks later, the nude mice were assessed for lung and liver metastases. Human colon cancer SW620 cells were used to study the effect of cathelicidin on cell migration and cytoskeleton. Incubation of SW620 cells with cathelicidin dose-dependently reduced cell migration, disrupted cytoskeletal structure, and reduced ßIII-tubulin (TUBB3) mRNA expression. The addition of the P2RX7 antagonist KN62, but not the FPRL1 antagonist WRW4, prevented the LL-37-mediated inhibition of cell migration and TUBB3 mRNA expression. The CAMP-HA-AAV-overexpressing group showed significantly reduced human CK20 protein (by 60%) and TUBB3 mRNA expression (by 40%) in the lungs and liver of the HT-29-loaded nude mice, compared to the HA-AAV control group. Intraperitoneal injection of KN62 reversed the CAMP-HA-AAV-mediated inhibition of human CK20 and TUBB3 expression in the lungs and liver of HT-29-loaded nude mice. In conclusion, cathelicidin inhibits colon cancer metastasis via a P2RX7-dependent pathway.

Ceragenin CSA13 Reduces Clostridium difficile Infection in Mice by Modulating the Intestinal Microbiome and Metabolites.

BACKGROUND & AIMS: Clostridium difficile induces intestinal inflammation by releasing toxins A and B. The antimicrobial compound cationic steroid antimicrobial 13 (CSA13) has been developed for treating gastrointestinal infections. The CSA13-Eudragit formulation can be given orally and releases CSA13 in the terminal ileum and colon. We investigated whether this form of CSA13 reduces C difficile infection (CDI) in mice. METHODS: C57BL/6J mice were infected with C difficile on day 0, followed by subcutaneous administration of pure CSA13 or oral administration of CSA13-Eudragit (10 mg/kg/d for 10 days). Some mice were given intraperitoneal vancomycin (50 mg/kg daily) on days 0-4 and relapse was measured after antibiotic withdrawal. The mice were monitored until day 20; colon and fecal samples were collected on day 3 for analysis. Blood samples were collected for flow cytometry analyses. Fecal pellets were collected each day from mice injected with CSA13 and analyzed by high-performance liquid chromatography or 16S sequencing; feces were also homogenized in phosphate-buffered saline and fed to mice with CDI via gavage. RESULTS: CDI of mice caused 60% mortality, significant bodyweight loss, and colonic damage 3 days after infection; these events were prevented by subcutaneous injection of CSA13 or oral administration CSA13-Eudragit. There was reduced relapse of CDI after administration of CSA13 was stopped. Levels of CSA13 in feces from mice given CSA13-Eudragit were significantly higher than those of mice given subcutaneous CSA13. Subcutaneous and oral CSA13 each significantly increased the abundance of Peptostreptococcaceae bacteria and reduced the abundance of C difficile in fecal samples of mice. When feces from mice with CDI and given CSA13 were fed to mice with CDI that had not received CSA13, the recipient mice had significantly increased rates of survival. CSA13 reduced fecal levels of inflammatory metabolites (endocannabinoids) and increased fecal levels of 4 protective metabolites (ie, citrulline, 3-aminoisobutyric acid, retinol, and ursodeoxycholic acid) in mice with CDI. Oral administration of these CSA13-dependent protective metabolites reduced the severity of CDI. CONCLUSIONS: In studies of mice, we found the CSA13-Eudragit formulation to be effective in eradicating CDI by modulating the intestinal microbiota and metabolites.

Fidaxomicin and OP-1118 Inhibit Clostridium difficile Toxin A- and B-Mediated Inflammatory Responses via Inhibition of NF-κB Activity.

Clostridium difficile causes diarrhea and colitis by releasing toxin A and toxin B. In the human colon, both toxins cause intestinal inflammation and stimulate tumor necrosis factor alpha (TNF-α) expression via the activation of NF-κB. It is well established that the macrolide antibiotic fidaxomicin is associated with reduced relapses of C. difficile infection. We showed that fidaxomicin and its primary metabolite OP-1118 significantly inhibited toxin A-mediated intestinal inflammation in mice in vivo and toxin A-induced cell rounding in vitro We aim to determine whether fidaxomicin and OP-1118 possess anti-inflammatory effects against toxin A and toxin B in the human colon and examine the mechanism of this response. We used fresh human colonic explants, NCM460 human colonic epithelial cells, and RAW264.7 mouse macrophages to study the mechanism of the activity of fidaxomicin and OP-1118 against toxin A- and B-mediated cytokine expression and apoptosis. Fidaxomicin and OP-1118 dose-dependently inhibited toxin A- and B-induced TNF-α and interleukin-1ß (IL-1ß) mRNA expression and histological damage in human colonic explants. Fidaxomicin and OP-1118 inhibited toxin A-mediated NF-κB phosphorylation in human and mouse intestinal mucosae. Fidaxomicin and OP-1118 also inhibited toxin A-mediated NF-κB phosphorylation and TNF-α expression in macrophages, which was reversed by the NF-κB activator phorbol myristate acetate (PMA). Fidaxomicin and OP-1118 prevented toxin A- and B-mediated apoptosis in NCM460 cells, which was reversed by the addition of PMA. PMA reversed the cytoprotective effect of fidaxomicin and OP-1118 in toxin-exposed human colonic explants. Fidaxomicin and OP-1118 inhibit C. difficile toxin A- and B-mediated inflammatory responses, NF-κB phosphorylation, and tissue damage in the human colon.

CSA13 inhibits colitis-associated intestinal fibrosis via a formyl peptide receptor like-1 mediated HMG-CoA reductase pathway.

Many Crohn's disease (CD) patients develop intestinal strictures, which are difficult to prevent and treat. Cationic steroid antimicrobial 13 (CSA13) shares cationic nature and antimicrobial function with antimicrobial peptide cathelicidin. As many functions of cathelicidin are mediated through formyl peptide receptor-like 1 (FPRL1), we hypothesize that CSA13 mediates anti-fibrogenic effects via FPRL1. Human intestinal biopsies were used in clinical data analysis. Chronic trinitrobenzene sulfonic acid (TNBS) colitis-associated intestinal fibrosis mouse model with the administration of CSA13 was used. Colonic FPRL1 mRNA expression was positively correlated with the histology scores of inflammatory bowel disease patients. In CD patients, colonic FPRL1 mRNA was positively correlated with intestinal stricture. CSA13 administration ameliorated intestinal fibrosis without influencing intestinal microbiota. Inhibition of FPRL1, but not suppression of intestinal microbiota, reversed these protective effects of CSA13. Metabolomic analysis indicated increased fecal mevalonate levels in the TNBS-treated mice, which were reduced by the CSA13 administration. CSA13 inhibited colonic HMG-CoA reductase activity in an FPRL1-dependent manner. Mevalonate reversed the anti-fibrogenic effect of CSA13. The increased colonic FPRL1 expression is associated with severe mucosal disease activity and intestinal stricture. CSA13 inhibits intestinal fibrosis via FPRL1-dependent modulation of HMG-CoA reductase pathway.

Circulating cathelicidin levels correlate with mucosal disease activity in ulcerative colitis, risk of intestinal stricture in Crohn's disease, and clinical prognosis in inflammatory bowel disease.

BACKGROUND: Cathelicidin (LL-37) is an antimicrobial peptide known to be associated with various autoimmune diseases. We attempt to determine if cathelicidin can accurately reflect IBD disease activity. We hypothesize that serum cathelicidin correlates with mucosal disease activity, stricture, and clinical prognosis of IBD patients. METHODS: Serum samples were collected from two separate cohorts of patients at the University of California, Los Angeles. Cohort 1 consisted of 50 control, 23 UC, and 28 CD patients. Cohort 2 consisted of 20 control, 57 UC, and 67 CD patients. LL-37 levels were determined by ELISA. Data from both cohorts were combined for calculation of accuracies in indicating mucosal disease activity, relative risks of stricture, and odds ratios of predicting disease development. RESULTS: Serum cathelicidin levels were inversely correlated with Partial Mayo Scores of UC patients and Harvey-Bradshaw Indices of CD patients. Among IBD patients with moderate or severe initial disease activity, the patients with high initial LL-37 levels had significantly better recovery than the patients with low initial LL-37 levels after 6-18 months, suggesting that high LL-37 levels correlate with good prognosis. Co-evaluation of LL-37 and CRP levels was more accurate than CRP alone or LL-37 alone in the correlation with Mayo Endoscopic Score of UC patients. Low LL-37 levels indicated a significantly elevated risk of intestinal stricture in CD patients. CONCLUSION: Co-evaluation of LL-37 and CRP can indicate mucosal disease activity in UC patients. LL-37 can predict future clinical activity in IBD patients and indicate risk of intestinal stricture in CD patients.

Probiotic Saccharomyces boulardii CNCM I-745 prevents outbreak-associated Clostridium difficile-associated cecal inflammation in hamsters.

C. difficile infection (CDI) is a common debilitating nosocomial infection associated with high mortality. Several CDI outbreaks have been attributed to ribotypes 027, 017, and 078. Clinical and experimental evidence indicates that the nonpathogenic yeast Saccharomyces boulardii CNCM I-745 (S.b) is effective for the prevention of CDI. However, there is no current evidence suggesting this probiotic can protect from CDI caused by outbreak-associated strains. We used established hamster models infected with outbreak-associated C. difficile strains to determine whether oral administration of live or heat-inactivated S.b can prevent cecal tissue damage and inflammation. Hamsters infected with C. difficile strain VPI10463 (ribotype 087) and outbreak-associated strains ribotype 017, 027, and 078 developed severe cecal inflammation with mucosal damage, neutrophil infiltration, edema, increased NF-κB phosphorylation, and increased proinflammatory cytokine TNFα protein expression. Oral gavage of live, but not heated, S.b starting 5 days before C. difficile infection significantly reduced cecal tissue damage, NF-κB phosphorylation, and TNFα protein expression caused by infection with all strains. Moreover, S.b-conditioned medium reduced cell rounding caused by filtered supernatants from all C. difficile strains. S.b-conditioned medium also inhibited toxin A- and B-mediated actin cytoskeleton disruption. S.b is effective in preventing C. difficile infection by outbreak-associated via inhibition of the cytotoxic effects of C. difficile toxins.

Substance P mediates pro-inflammatory cytokine release form mesenteric adipocytes in Inflammatory Bowel Disease patients.

BACKGROUND & AIMS: Substance P (SP), neurokinin-1 receptors (NK-1Rs) are expressed in mesenteric preadipocytes and SP binding activates proinflammatory signalling in these cells. We evaluated the expression levels of SP (Tac-1), NK-1R (Tacr-1), and NK-2R (Tacr-2) mRNA in preadipocytes isolated from patients with Inflammatory Bowel Disease (IBD) and examined their responsiveness to SP compared to control human mesenteric preadipocytes. The Aim of our study is to investigate the effects of the neuropeptide SP on cytokine expression in preadipocytes of IBD vs control patients and evaluate the potential effects of these cells on IBD pathophysiology via SP-NK-R interactions. METHODS: Mesenteric fat was collected from control, Ulcerative colitis (UC) and Crohn's disease (CD) patients (n=10-11 per group). Preadipocytes were isolated, expanded in culture and exposed to substance P. Colon biopsies were obtained from control and IBD patients. RESULTS: Tacr-1 and -2 mRNA were increased in IBD preadipocytes compared to controls, while Tac-1 mRNA was increased only in UC preadipocytes. SP differentially regulated the expression of inflammatory mediators in IBD preadipocytes compared to controls. Disease-dependent responses to SP were also observed between UC and CD preadipocytes. IL-17A mRNA expression and release increased after SP treatment in both CD and UC preadipocytes, while IL-17RA mRNA increased in colon biopsies from IBD patients. CONCLUSIONS: Preadipocyte SP-NK-1R interactions during IBD may participate in IBD pathophysiology. The ability of human preadipocytes to release IL-17A in response to SP together with increased IL-17A receptor in IBD colon opens the possibility of a fat-colonic mucosa inflammatory loop that may be active during IBD.

Anti-fibrogenic effects of the anti-microbial peptide cathelicidin in murine colitis-associated fibrosis.

BACKGROUND AND AIMS: Cathelicidin (LL-37 in human and mCRAMP in mice) represents a family of endogenous antimicrobial peptides with anti-inflammatory effects. LL-37 also suppresses collagen synthesis, an important fibrotic response, in dermal fibroblasts. Here we determined whether exogenous cathelicidin administration modulates intestinal fibrosis in two animal models of intestinal inflammation and in human colonic fibroblasts. METHODS: C57BL/6J mice (n=6 per group) were administered intracolonically with a trinitrobenzene sulphonic acid (TNBS) enema to induce chronic (6-7 weeks) colitis with fibrosis. mCRAMP peptide (5 mg/kg every 3 day, week 5-7) or cathelicidin gene (Camp)-expressing lentivirus (107 infectious units week 4) were administered intracolonically or intravenously, respectively. 129Sv/J mice were infected with Salmonella typhimurium orally to induce cecal inflammation with fibrosis. Camp expressing lentivirus (107 infectious units day 11) was administered intravenously. RESULTS: TNBS-induced chronic colitis was associated with increased colonic collagen (col1a2) mRNA expression. Intracolonic cathelicidin (mCRAMP peptide) administration or intravenous delivery of lentivirus-overexpressing cathelicidin gene significantly reduced colonic col1a2 mRNA expression in TNBS-exposed mice, compared to vehicle administration. Salmonella infection also caused increased cecal inflammation associated with collagen (col1a2) mRNA expression that was prevented by intravenous delivery of Camp-expressing lentivirus. Exposure of human primary intestinal fibroblasts and human colonic CCD-18Co fibroblasts to transforming growth factor-beta1 (TGF-beta1) and/or insulin-like growth factor 1 induced collagen protein and mRNA expression, that was reduced by LL-37 (3-5 µM) through a MAP kinase-dependent mechanism. CONCLUSION: Cathelicidin can reverse intestinal fibrosis by directly inhibiting collagen synthesis in colonic fibroblasts.

Cathelicidin suppresses colon cancer development by inhibition of cancer associated fibroblasts.

BACKGROUND: Cathelicidin (LL-37 in humans and mCRAMP in mice) represents a family of endogenous antimicrobial and anti-inflammatory peptides. Cancer-associated fibroblasts can promote the proliferation of colon cancer cells and growth of colon cancer tumors. METHODS: We examined the role of cathelicidin in the development of colon cancer, using subcutaneous human HT-29 colon-cancer-cell-derived tumor model in nude mice and azoxymethane- and dextran sulfate-mediated colon cancer model in C57BL/6 mice. We also determined the indirect antitumoral mechanism of cathelicidin via the inhibition of epithelial-mesenchymal transition (EMT) of colon cancer cells and fibroblast-supported colon cancer cell proliferation. RESULTS: Intravenous administration of cathelicidin expressing adeno-associated virus significantly reduced the size of tumors, tumor-derived collagen expression, and tumor-derived fibroblast expression in HT-29-derived subcutaneous tumors in nude mice. Enema administration of the mouse cathelicidin peptide significantly reduced the size and number of colonic tumors in azoxymethane- and dextran sulfate-treated mice without inducing apoptosis in tumors and the adjacent normal colonic tissues. Cathelicidin inhibited the collagen expression and vimentin-positive fibroblast expression in colonic tumors. Cathelicidin did not directly affect HT-29 cell viability, but did significantly reduce tumor growth factor-ß1-induced EMT of colon cancer cells. Media conditioned by the human colonic CCD-18Co fibroblasts promoted human colon cancer HT-29 cell proliferation. Cathelicidin pretreatment inhibited colon cancer cell proliferation mediated by media conditioned by human colonic CCD-18Co fibroblasts. Cathelicidin disrupted tubulin distribution in colonic fibroblasts. Disruption of tubulin in fibroblasts reduced fibroblast-supported colon cancer cell proliferation. CONCLUSION: Cathelicidin effectively inhibits colon cancer development by interfering with EMT and fibroblast-supported colon cancer cell proliferation.

Endothelin-converting enzyme 1 and ß-arrestins exert spatiotemporal control of substance P-induced inflammatory signals.

Although the intracellular trafficking of G protein-coupled receptors controls specific signaling events, it is unclear how the spatiotemporal control of signaling contributes to complex pathophysiological processes such as inflammation. By using bioluminescence resonance energy transfer and superresolution microscopy, we found that substance P (SP) induces the association of the neurokinin 1 receptor (NK1R) with two classes of proteins that regulate SP signaling from plasma and endosomal membranes: the scaffolding proteins ß-arrestin (ßARRs) 1 and 2 and the transmembrane metallopeptidases ECE-1c and ECE-1d. In HEK293 cells and non-transformed human colonocytes, we observed that G protein-coupled receptor kinase 2 and ßARR1/2 terminate plasma membrane Ca(2+) signaling and initiate receptor trafficking to endosomes that is necessary for sustained activation of ERKs in the nucleus. ßARRs deliver the SP-NK1R endosomes, where ECE-1 associates with the complex, degrades SP, and allows the NK1R, freed from ßARRs, to recycle. Thus, both ECE-1 and ßARRs mediate the resensitization of NK1R Ca(2+) signaling at the plasma membrane. Sustained exposure of colonocytes to SP activates NF-κB and stimulates IL-8 secretion. This proinflammatory signaling is unaffected by inhibition of the endosomal ERK pathway but is suppressed by ECE-1 inhibition or ßARR2 knockdown. Inhibition of protein phosphatase 2A, which also contributes to sustained NK1R signaling at the plasma membrane, similarly attenuates IL-8 secretion. Thus, the primary function of ßARRs and ECE-1 in SP-dependent inflammatory signaling is to promote resensitization, which allows the sustained NK1R signaling from the plasma membrane that drives inflammation.

Fidaxomicin inhibits Clostridium difficile toxin A-mediated enteritis in the mouse ileum.

Clostridium difficile infection (CDI) is a common, debilitating infection with high morbidity and mortality. C. difficile causes diarrhea and intestinal inflammation by releasing two toxins, toxin A and toxin B. The macrolide antibiotic fidaxomicin was recently shown to be effective in treating CDI, and its beneficial effect was associated with fewer recurrent infections in CDI patients. Since other macrolides possess anti-inflammatory properties, we examined the possibility that fidaxomicin alters C. difficile toxin A-induced ileal inflammation in mice. The ileal loops of anesthetized mice were injected with fidaxomicin (5, 10, or 20 µM), and after 30 min, the loops were injected with purified C. difficile toxin A or phosphate-buffered saline alone. Four hours after toxin A administration, ileal tissues were processed for histological evaluation (epithelial cell damage, neutrophil infiltration, congestion, and edema) and cytokine measurements. C. difficile toxin A caused histologic damage, evidenced by increased mean histologic score and ileal interleukin-1ß (IL-1ß) protein and mRNA expression. Treatment with fidaxomicin (20 µM) or its primary metabolite, OP-1118 (120 µM), significantly inhibited toxin A-mediated histologic damage and reduced the mean histology score and ileal IL-1ß protein and mRNA expression. Both fidaxomicin and OP-1118 reduced toxin A-induced cell rounding in human colonic CCD-18Co fibroblasts. Treatment of ileal loops with vancomycin (20 µM) and metronidazole (20 µM) did not alter toxin A-induced histologic damage and IL-1ß protein expression. In addition to its well known antibacterial effects against C. difficile, fidaxomicin may possess anti-inflammatory activity directed against the intestinal effects of C. difficile toxins.